Bipolar trans carotenoid salts and their uses

ABSTRACT

The invention relates to trans carotenoid salt compounds, methods for making them, methods for solubilizing them and uses thereof. These compounds are useful in improving diffusivity of oxygen between red blood cells and body tissues in mammals including humans.

The invention relates to bipolar trans carotenoid salt compounds,methods of solubilizing them, methods for making them, and methods ofusing them. These bipolar trans carotenoid salts (BTCS) compounds areuseful in improving diffusivity of oxygen between red blood-cells andbody tissues in mammals including humans.

BACKGROUND OF THE INVENTION

Carotenoids are a class of hydrocarbons consisting of isoprenoid unitsjoined in such a manner that their arrangement is reversed at the centerof the molecule. The backbone (skeleton) of the molecule consists ofconjugated carbon-carbon double and single bonds, and can also havependant groups. Although it was once thought that the skeleton of acarotenoid contained 40 carbons, it has been long recognized thatcarotenoids can also have carbon skeletons containing fewer than 40carbon atoms. The 4 single bonds that surround a carbon-carbon doublebond all lie in the same plane. If the pendant groups are on the sameside of the carbon-carbon double bond, the groups are designated as cis;if they are on opposite side of the carbon-carbon bond, they aredesignated as trans. Because of the large number of double bonds, thereare extensive possibilities for geometrical (cis/trans) isomerism ofcarotenoids, and isomerization occurs readily in solution. A recentseries of books is an excellent reference to many of the properties,etc. of carotenoids (“Carotenoids”, edited by G. Britton, S.Liaaen-Jensen and H. Pfander, Birkhauser Verlag, Basel, 1995 herebyincorporated by reference in its entirety).

Many carotenoids are nonpolar and, thus, are insoluble in water. Thesecompounds are extremely hydrophobic which makes their formulation forbiological uses difficult because, in order to solubilize them, one mustuse an organic solvent rather than an aqueous solvent. Other carotenoidsare monopolar, and have characteristics of surfactants (a hydrophobicportion and a hydrophilic polar group). As such, these compounds areattracted to the surface of an aqueous solution rather than dissolvingin the bulk liquid. A few natural bipolar carotenoid compounds exist,and these compounds contain a central hydrophobic portion as well as twopolar groups, one on each end of the molecule. It has been reported(“Carotenoids”, Vol. 1A, p. 283) that carotenoid sulphates have“significant solubility in water of up to 0.4 mg/ml”. Other carotenoidsthat might be thought of as bipolar are also not very soluble in water.These include dialdehydes and diketones. A di-pyridine salt of crocetinhas also been reported, but its solubility in water is less than 1 mg/mlat room temperature. Other examples of bipolar carotenoids are crocetinand crocin (both found in the spice saffron). However, crocetin is onlysparingly soluble in water. In fact, of all of the bipolar carotenoids,only crocin displays significant solubility in water.

U.S. Pat. Nos. 4,176,179; 4,070,460; 4,046,880; 4,038,144; 4,009,270;3,975,519; 3,965,261; 3,853,933; and 3,788,468 relate to various uses ofcrocetin.

U.S. Pat. No. 5,107,030 relates to a method of making2,7-dimethyl-2,4,6-octatrienedial and derivatives thereof.

U.S. Pat. No. 6,060,511 relates to trans sodium crocetinate (TSC) andits uses. The TSC is made by reacting naturally occurring saffron withsodium hydroxide followed by extractions.

In Roy et al, Shock 10, 213-7. (1998), hemorrhaged rats (55% bloodvolume) were given a bolus of trans sodium crocetinate (TSC) after 10minutes, followed by saline after another 30 minutes. All of theTSC-treated animals lived, while all controls died. Whole-body oxygenconsumption increased in the TSC group, reaching 75% of normal restingvalue after about 15 minutes.

Laidig et al, J Am Chem. Soc. 120, 9394-9395 (1998), relates tocomputational modeling of TSC. A simulated TSC molecule was “hydrated”by surrounding it with water molecules. The hydrophobic ordering of thewater in the vicinity of the TSC made it easier for oxygen molecules todiffuse through the system. The computational increase in diffusivity of˜30% was consistent with results obtained in both in vitro and animalexperiments.

In Singer et al, Crit Care Med 28, 1968-72. (2000), TSC improvedhemodynamic status and prolonged rat survival in a rat model of acutehypoxia. Hypoxia was induced using a low oxygen concentration (10%) airmixture: after 10 minutes the animals were given either saline or TSC.Hypoxemia led to a reduction in blood flow, and an increase in basedeficit. Only 2 of 6 animals survived in the control group. The treatedgroup all survived with good hemodynamic stability for over two hours,with a slow decline thereafter.

SUMMARY OF THE INVENTION

The subject invention relates to bipolar trans carotenoid salts (BTCS)compounds and synthesis of such compounds having the structure:YZ-TCRO-ZY

where:

-   -   Y=a cation    -   Z=polar group which is associated with the cation, and    -   TCRO=trans carotenoid skeleton.

The subject invention also relates to individual BTCS compoundcompositions (including a TSC composition) wherein absorbency of thehighest peak (of an aqueous solution of the BTCS composition) whichoccurs in the visible wave length range divided by the absorbency of thepeak which occurs in the UV wave length range, is greater than 8.5,advantageously greater than 9, most advantageously greater than 9.5.

The invention also relates to a method of treating a variety of diseasescomprising administering to a mammal in need of treatment atherapeutically effective amount of a compound having the formula:YZ-TCRO-ZY

The invention also includes several methods of solubilizing andsynthesizing compounds having the formula:YZ-TCRO-ZY

The invention also relates to an inhaler for delivery of the compoundsof the invention.

DETAILED DESCRIPTION OF THE INVENTION

A new class of carotenoid and carotenoid related compounds has beendiscovered. These compounds are referred to as “bipolar trans carotenoidsalts” (BTCS).

Compounds of the Invention

The subject invention relates to a class of compounds, bipolar transcarotenoid salts, that permit the hydrophobic carotenoid or carotenoidrelated skeleton to dissolve in an aqueous solution, and methods formaking them. The cations of these salts can be a number of species, butadvantageously sodium or potassium (these are found in most biologicalsystems). Commonly owned U.S. Pat. No. 6,060,511, hereby incorporated byreference in its entirety, describes an extraction method for makingtrans sodium crocetinate, TSC (a BTCS) starting from saffron.

A general structure for a bipolar trans carotenoid salt is:YZ-TCRO-ZY

where:

-   -   Y (which can be the same or different at the two ends)=a cation,        preferably Na⁺ or K⁺ or Li⁺. Y is advantageously a monovalent        metal ion. Y can also be an organic cation, e.g., R₄N⁺, R₃S⁺,        where R is H, or C_(n)H_(2n+1) where n is 1-10, advantageously        1-6. For example, R can be methyl, ethyl, propyl or butyl.    -   Z (which can be the same or different at the two ends)=polar        group which is associated with the cation. Optionally including        the terminal carbon on the carotenoid (or carotenoid related        compound), this group can be a carboxyl (COO⁻) group or a CO        group. This group can also be a sulfate group (OSO₃ ⁻) or a        monophosphate group (OPO₃ ⁻), (OP(OH)O₂ ⁻), a diphosphate group,        triphosphate or combinations thereof.    -   TCRO=trans carotenoid or carotenoid related skeleton        (advantageously less than 100 carbons) which is linear, has        pendant groups (defined below), and typically comprises        “conjugated” or alternating carbon-carbon double and single        bonds (in one embodiment, the TCRO is not fully conjugated as in        a lycopene). The pendant groups are typically methyl groups but        can be other groups as discussed below. In an advantageous        embodiment, the units of the skeleton are joined in such a        manner that their arrangement is reversed at the center of the        molecule. The 4 single bonds that surround a carbon-carbon        double bond all lie in the same plane. If the pendant groups are        on the same side of the carbon-carbon double bond, the groups        are designated as cis; if they are on the opposite side of the        carbon-carbon bond, they are designated as trans. The compounds        of the subject invention are trans. The cis isomer typically is        a detriment—and results in the diffusivity not being increased.        In one embodiment, a trans isomer can be utilized where the        skeleton remains linear.

Examples of trans carotenoid or carotenoid related skeletons are:

where pendant groups X (which can be the same or different) are hydrogen(H) atoms, or a linear or branched group having 10 or less carbons,advantageously 4 or less, (optionally containing a halogen), or ahalogen. Examples of X are a methyl group (CH₃), an ethyl group (C₂H₅),a halogen-containing alkyl group (C1-C10) such as CH₂Cl, or a halogensuch as Cl or Br. The pendant X groups can be the same or different butthe X groups utilized must maintain the skeleton as linear.

Although many carotenoids exist in nature, carotenoid salts do not.Commonly owned U.S. Pat. No. 6,060,511 relates to trans sodiumcrocetinate (TSC). The TSC was made by reacting naturally occurringsaffron with sodium hydroxide followed by extractions that selectedprimarily for the trans isomer.

The presence of the cis and trans isomers of BTCS can be determined bylooking at the ultraviolet-visible spectrum for the carotenoid sampledissolved in an aqueous solution. Given the spectrum, the value of theabsorbency of the highest peak which occurs in the visible wave lengthrange of 416 to 423 nm (the number depending on the solvent used) isdivided by the absorbency of the peak which occurs in the UV wave lengthrange of 250 to 256 nm, can be used to determine the purity level of thetrans isomer. When the BTCS is dissolved in water, the highest visiblewave length range peak will be at about 421 nm and the UV wave lengthrange peak will be at about 254 nm. According to M. Craw and C. Lambert,Photochemistry and Photobiology, Vol. 38 (2), 241-243 (1983) herebyincorporated by reference in its entirety, the result of the calculation(in that case crocetin was analysed) was 3.1, which increased to 6.6after purification.

Performing the Craw and Lambert analysis on the trans sodium crocetin ofcommonly owned U.S. Pat. No. 6,060,511 (TSC made by reacting naturallyoccurring saffron with sodium hydroxide followed by extractions whichselected primarily for the trans isomer), the value obtained istypically around 7-7.5 (a value of 8.4 was observed once). Performingthat test on the synthetic TSC of the subject invention, that ratio istypically greater than 8.5 (e.g. 8.5 to 10), advantageously greater than9 (e.g. 9-10), most advantageously greater than 9.5. For the TSCsynthesized according to the improved method of Example 5, the ratio istypically greater than 9.5 (e.g. 9.5-12). The synthesized material is a“purer” or highly purified trans isomer.

It has been found, recently, that TSC has an aqueous solubility ofgreater than 10 mg/ml at room temperature, which is remarkable for amolecule containing such a long, hydrophobic portion. TSC has also beenfound to increase the diffusivity of oxygen through liquids.

U.S. Pat. No. 6,060,511 describes an extraction method for making TSCstarting from saffron; however, other bipolar carotenoid salts cannot bemade using that same procedure since the use of saffron allows only asingle carotenoid skeleton to be incorporated into the salt.

The invention disclosed herein allows the synthesis of a whole class ofcompounds: bipolar trans carotenoid salts which contain variouscarotenoid or carotenoid related skeletons. Such compounds are solublein aqueous solutions and have advantageous biological uses, such ascausing an increase in oxygen utilization. It is believed that thisincrease is a result of the ability of the hydrophobic portion (theskeleton) of the bipolar trans carotenoid salt to affect the bonding ofnearby water molecules. This, in effect, allows the oxygen molecule todiffuse faster while in this vicinity.

Solubilizing the Compounds and Compositions of the Invention

The invention allows for the dissolution of a trans carotenoid orcarotenoid related skeleton molecule in aqueous solutions. The novelmethods of dissolution are related below. The methods apply to anybipolar trans carotenoid salt and composition thereof.

BTCS-Containing Saline Infusion Solutions

Large volumes (as much as 3 times the estimated blood loss) of isotonicsaline (also called normal saline) are infused as a treatment forhemorrhagic shock. The isotonic saline contains 9 g NaCl per liter ofwater so as not to disturb the ionic strength of the plasma once it isinfused into the body. Adding TSC to the saline has been shown to resultin a superior infusion fluid, however, one cannot simply mix TSC powderwith the saline to make such a solution. About 50% of the TSC dissolvesin normal saline no matter how much TSC is added (up to severalmilligrams per ml), which means that undissolved particles of TSC arestill present. In order to prevent that, a stock solution can be made byadding more than twice the amount of TSC needed and then centrifugingout the particles that do not dissolve. The actual composition of thestock solution can be verified using UV-visible spectroscopy. This stocksolution can be added to normal saline and the TSC remains dissolved.

This method can be used to dissolve a BTCS in other types of sodiumchloride solutions, as well as in solutions of other salts such as KCl,Na₂SO₄, lactate, etc. Several, eg 1-3 mg/ml, can be put into solution inthis manner.

Dilute Solution of Sodium Carbonate Dissolves BTCS

A BTCS such as TSC dissolves in very dilute sodium carbonate solutions.A dilute, eg 0.00001-0.001M, solution of sodium carbonate can be added,dropwise, to deionized water until the pH is 8.0 (the pH of deionizedwater is usually 5-6). This only takes a few drops of the very dilutesodium carbonate per, say, 50 mls of deionized water. This sodiumcarbonate-deionized water solution is capable of completely dissolving alarge amount of TSC (greater than 10 mg/ml)—which is remarkableconsidering the hydrophobicity of the carotenoid portion of the BTCS.

A BTCS can be supplied as a powder along with a sterilized bottle of thesodium carbonate water. This concentrated solution can then be injecteddirectly (very small volumes of solutions having a lower ionic strengththan plasma can be injected), or the concentrated solution can be addedto normal saline and then injected. If TSC is dissolved in the sodiumcarbonate-water solvent and then more of the same solvent is added—theTSC stays in solution.

In another embodiment, sodium bicarbonate is used instead of sodiumcarbonate. Other salts which result in the deionized water having abasic pH can also be used.

Carotenoid skeleton concentrations of 5-10 mg/ml can be achieved withthis procedure.

Water Dissolves BTCS

Although TSC dissolves in water (tap, distilled, deionized), thesesolutions are only stable if the pH is adjusted so as to make thesolution basic. TSC is more soluble in deionized water (very few Na⁺ions present) than in normal water. A BTCS, such as TSC, will dissolvein just deionized water alone, but, if plain deionized water is added tothat solution, the TSC will precipitate out. A BTCS will dissolve injust deionized water alone, but additional deionized water may causeprecipitation of the BTCS if the pH is not adjusted to make it slightlybasic.

Other Methods of Solubilizing BTSC

The BTCS can be formulated in a delivery system that enhances delivery.See Formulations of the Compounds of the Invention below.

Synthesis of the Compounds of the Invention

Bipolar Trans Carotenoid Salts

Set forth below are the novel synthesis methods that can be used forsynthesizing bipolar trans carotenoid salts. There can be variations invarious steps of the synthesis that are obvious to one skilled in theart.

A. TSC Synthesis

Trans sodium crocetinate (TSC) can be synthesized by coupling asymmetrical C₁₀ dialdehyde containing conjugated carbon-carbon doublebonds (2,7-dimethylocta-2,4,6-triene-1,8-dial) with[3-carbomethoxy-2-buten-1-ylidene]triphenylphosphorane. This results inthe formation of a trans dimethyl ester of crocetin. This dimethyl esteris then converted to the final TSC product by saponification. Typically,saponification is accomplished by treating an ester with either aqueoussodium hydroxide or sodium hydroxide dissolved in THF (tetrahydrofuran);however, these methods did not give the best results in this case.Saponification can be accomplished very well, in this case, by reactingthe ester with an NaOH/methanol solution. After saponification, the TSCis recovered by drying in a vacuum.

The C₁₀ dialdehyde and the triphenylphosphorane reactants used in thissynthesis can be made via different routes. For example, the C₁₀dialdehyde was prepared starting with ethyl bromoacetate and furan usingWittig chemistry. Tiglic acid was the starting material for making thedesired phosphorane. Different lengths of carotenoid skeletons can bemade by joining together reactants of different lengths (for example aC₁₄ dialdehyde and triphenylphosphorane). This procedure results in theformation of different trans bipolar carotenoid salts. Alterations canalso be made so as to obtain different pendant groups (TSC has methylgroups for the pendant groups).

The TSC made in this manner is soluble in water (pH adjusted to 8.0 witha very dilute solution of sodium carbonate) at a level >10 mg/ml at roomtemperature. Other bipolar trans carotenoid salts are soluble at roomtemperature in water having a pH that is neutral or above. As usedherein, “soluble” means that amounts greater than 5 mg will dissolve perml of water at room temperature (as noted previously, carotenoidreferences state that 0.4 mg/ml is “highly significant solubility”—butthat is lower than the subject definition of solubility).

B. General Synthesis

Carotenoid or carotenoid related structures can be built up in thefollowing manner:

(3-carbomethoxy-2-buten-1-ylidene)triphenylphosphorane (or a relatedcompound when X is other than a methyl group) is a key precursor to addisoprenoid units (or isoprenoid related units) to both ends of asymmetrical carotenoid (or carotenoid related compound). This processcan be repeated infinitely. For example, dimethyl trans crocetinate canbe reduced to the corresponding symmetrically dialdehyde using thechemistry described above. This dialdehyde can be reacted with excess(3-carbomethoxy-2-buten-1-ylidene)triphenylphosphorane to give thecorresponding diester. This synthetic sequence can be repeated again andagain.

Improved Synthesis

2,7-Dimethyl-2,4,6-octatrienedial is a key intermediate toward thesynthesis of TSC. This key precursor has three double bonds and thusseveral isomers are possible. For TSC, the all trans isomer(E,E,E-isomer) is required. The general synthesis route involves an11-step synthesis with relatively low yields and poor selectivity inseveral steps (see Example 1). As a result, column chromatography isrequired to purify several intermediates along the way.

The improved synthesis route is much simpler (see the reaction schemebelow). The 3-step process as described in U.S. Pat. No. 5,107,030,hereby incorporated by reference in its entirety, gives a mixture ofgeometric isomers of the dialdehyde (U.S. Pat. No. 5,107,030 does notnote this mixture). In the method of the subject invention described inExample 1, 96-97% of the desired isomer (all trans or E,E,E-isomer) isobtained by several recrystallizations from methanol or ethyl acetate in59% yield.

The improved synthesis method of the subject invention involvesconverting the remaining isomeric mixture of dialdehydes into thedesired trans aldehyde (E,E,E) by isomerization with a sulfinic acid(RSO2H where R is C1 through C10 straight or branched alkyl group or anaryl group (a substituted phenyl group) such as para-toluenesulfinicacid, in an appropriate solvent such as 1,4-dioxane, tetrahydrofuran ordialkyl ether where the alkyl group is one or two of a C1 through C10straight or branched alkyl group. An additional 8% yield of the puredesired dialdehyde is obtained, raising the overall yield of the laststep from 59% to 67% yield. This yield improvement is important. Thisisomerization step can be incorporated into the third step of the methodof U.S. Pat. No. 5,107,030 to get a better yield.Improved Synthesis Route:

Two Undesired Isomers:

Isomerization of Undesired to Desired Dialdehyde:

Saponification can be accomplished by dissolving the diester in methanoland then adding a base such as NaOH (Y of the BTCS is then Na⁺).Alternatively, the diester can be dissolved in methanol alreadycontaining the base. The NaOH is typically aqueous (20-60% by wt.) butcan be solid. Alternatives to methanol for dissolving the diester areethanol, propanol and isopropanol. Saponification can be carried out invarious ways commercially. A one or two phase system (one organic andone aqueous phase) can be used.

Trans crocetin can also be synthesized according to the methodsdescribed above.

In addition, as has been reported for TSC, such BTCS compounds increasethe diffusivity of oxygen through water (this will also depend on thenature of the hydrophobic portion incorporated into the final productsuch as carbon chain length) since it is believed that the hydrophobicinteractions of the carotenoid skeleton with water result in theincreased diffusivity).

Formulations of the Compounds of the Invention

A concentrated solution of a bipolar trans carotenoid salt can be made,as described previously, by dissolving it in a very dilute solution ofsodium carbonate. The resulting mixture can then be used in that manner,or can be diluted further with normal saline or other aqueous solvents.In addition, solutions of a bipolar trans carotenoid salt can be made bydissolving the bipolar trans carotenoid salt directly in a salt solutionand then getting rid of any material that does not dissolve.

The bipolar trans carotenoid salts are stable in a dry form at roomtemperature, and can be stored for long periods. Advantageously, aformulation of such salts, if given orally, is absorbed in the gut,rather than the stomach.

Although the compounds of the invention can be administered alone, theycan be administered as part of a pharmaceutical formulation. Suchformulations can include pharmaceutically acceptable carriers known tothose skilled in the art as well as other therapeutic agents-see below.Advantageously, the formulation does not include a compound thatinhibits the ability of the compounds of the invention to improvediffusivity of oxygen.

Appropriate dosages of the compounds and compositions of the inventionwill depend on the severity of the condition being treated. For a doseto be “therapeutically effective”, it must have the desired effect, i.e.increase the diffusivity of oxygen. This in turn, will causeoxygen-related parameters to return towards normal values.

Administration can be by any suitable route including oral, nasal,topical, parenteral (including subcutaneous, intramuscular, intravenous,intradermal and intraosseus), vaginal or rectal. The preferred route ofadministration will depend on the circumstances. An inhalation route isadvantageous for treatment in emergency situations, where it isnecessary for the BTCS to enter the bloodstream very quickly. Theformulations thus include those suitable for administration through suchroutes (liquid or powder to be nebulized). It will be appreciated thatthe preferred route may vary, for example, with the condition and age ofthe patient. The formulations can conveniently be presented in unitdosage form, e.g., tablets and sustained release capsules, and can beprepared and administered by methods known in the art of pharmacy. Theformulation can be for immediate, or slow or controlled release of theBTCS. See for example, the controlled release formulation of WO 99/15150hereby incorporated by reference its entirety.

Formulations of the present invention suitable for oral administrationcan be presented as discrete units such as pills, capsules, cachets ortablets, as powder or granules, or as a solution, suspension oremulsion. Formulations suitable for oral administration further includelozenges, pastilles, and inhalation mists administered in a suitablebase or liquid carrier. Formulations for topical administration to theskin can be presented as ointments, creams, gels, and pastes comprisingthe active agent and a pharmaceutically acceptable carrier or in atransdermal patch.

Formulations suitable for nasal administration wherein the carrier is asolid include powders of a particular size that can be administered byrapid inhalation through the nasal passage. Suitable formulationswherein the carrier is a liquid can be administered, for example as anasal spray or drops.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions that can contain antioxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient, and aqueous and nonaqueoussterile suspensions which can include suspending agents and thickeningagents. The formulations can be presented in unit or multi-dosecontainers, for example sealed ampules and vials, and can belyophilized, requiring only the addition of the sterile liquid carriersuch as water for injection immediately prior to use. Injectionsolutions and suspensions can be prepared from sterile powders, granulesand tablets.

Uses of the Compounds and Compositions of the Invention

A wide variety of conditions are controlled or are mediated by deliveryof oxygen to body tissues. The compounds and compositions of the subjectinvention can be used in the same pharmaceutical applications describedfor crocetin in the same effective amounts; see U.S. Pat. Nos.4,176,179; 4,070,460; 4,046,880; 4,038,144; 4,009,270; 3,975,519;3,965,261; 3,853,933; and 3,788,468 each of which is hereby incorporatedby reference in its entirety.

TSC has been shown to increase the diffusivity of oxygen through aqueoussolutions by about 30%. Thus, the compounds of the invention are usefulfor treating diseases/conditions which are characterized by low oxygen(hypoxia) such as respiratory diseases, hemorrhagic shock andcardiovascular diseases, atherosclerosis, emphysema, asthma,hypertension, cerebral edema, papillomas, spinal cord injuries, amongothers. Other bipolar trans carotenoid salts have similar properties.Such compounds can also be used in conjunction with other methodscommonly suggested for increasing oxygen utilization in the body, suchas oxygen therapy and the use of hemoglobins or fluorocarbons.

In one embodiment of the invention, a BTCS is administered to thepatient while administering oxygen. Alternatively, hemoglobins orfluorocarbons and a BTSC can be given together. In these cases, anadditive effect is realized.

The minimum dosage needed for treatment for any of these salts is thatat which the diffusivity of oxygen increases. The effective dosage ofthe compounds of the inventions will depend upon the condition treated,the severity of the condition, the stage and individual characteristicsof each mammalian patient addressed. Dosage will vary, however, fromabout 0.001 mg of active compound per kg of body weight up to about 500mg per kg, and advantageously from about 0.01-30 mg/kg of body weight.IV administration is advantageous but other routes of injection can alsobe used such as intramuscular, subcutaneous or via inhalation. Oraladministration can also be used as can transdermal delivery orintraosseus delivery.

Respiratory Disorders

Bipolar trans carotenoid salts can be used to treat respiratorydisorders. These are described as conditions in which the arterialpartial pressure of oxygen is reduced, such as value of 60 to 70 mm Hgrather than the normal value of 90-100 mm Hg. Such diseases areemphysema, acute respiratory distress syndrome (ARDS) or chronicobstructive pulmonary disease (COPD).

TSC increases the value of the partial pressure of oxygen in the bloodwhen it is low (this is a symptom of emphysema, ARDS and COPD).Increasing the partial pressure of oxygen in the blood relieves many ofthe symptoms of emphysema, ARDS and COPD. TSC does not cure the cause ofthe disease, but relieves the oxidative distress and damage resultingfrom that underlying cause.

Hemorrhagic Shock

Hemorrhagic shock is marked by a decrease in oxygen consumption. Bipolartrans carotenoid salts increase the body's oxygen consumption by causingmore oxygen to diffuse from the red blood cells to the tissues. TSC hasbeen shown to increase the oxygen consumption of rats undergoinghemorrhagic shock, and has also been shown to offset other symptoms ofshock. The compounds of the invention cause the low blood pressure toincrease, reduce the increased heart rate, and reverse the bloodacidosis that develops during shock. The compounds of the invention alsoreduce organ damage subsequent to hemorrhagic shock.

The compounds of the invention can be used for hemorrhagic shock byadministering them by inhalation, injecting them, or by adding them to astandard resuscitation fluid (Ringer's lactate or normal saline).

Cardiovascular Disease

In western culture, the leading cause of death is ischemic heartdisease. Death may result from either a gradual deterioration of theability of the heart to contract or, frequently, a sudden stoppage.Sudden cardiac death (SCD) covers the time period beginning 60 secondsafter symptoms begin to 24 hours later. These deaths are usually aconsequence of acute coronary occlusion (blockage) or of ventricularfibrillation (which can result from the occlusion).

Myocardial ischemia exists when there is an insufficient supply ofoxygen to the cardiac muscle. When coronary blood flow is extremely low,cardiac muscle cannot function and dies. That area of muscle is said tobe infarcted. Most often, diminished coronary blood flow is caused byatherosclerosis that occurs in the coronary arteries. Ischemia resultsin impaired mechanical and electrical performance and muscle cellinjury, which may lead to a lethal arrhythmia, called ventricularfibrillation (VF). In ventricular fibrillation, the electrical activityof the ventricles of the heart is chaotic and results in anelectrocardiogram with an erratic rhythm and no recognizable patterns.Ventricular fibrillation occurs frequently with myocardial ischemia andinfarction and is nearly always the cause of sudden cardiac death.Bipolar trans carotenoid salts are beneficial in treating myocardialischemia. Atherosclerosis, which is frequently a precursor to myocardialinfarction, can also be treated with these salts.

Ischemia

Bipolar trans carotenoid salts are also beneficial in treating otherforms of ischemia (insufficient blood flow to tissues or organs) such askidney, liver, spinal cord, and brain ischemia including stroke.

Hypertension

Hypertension, or high blood pressure, is frequently associated withcardiovascular disease. The compounds of the invention can be used toreduce blood pressure.

Performance Enhancement

BTCS enhance aerobic metabolism, increasing oxygen consumption levelsduring walking, running, lifting, etc. Endurance is also increased.

Traumatic Brain Injury

Hypoxia following traumatic brain injury results in increased braindamage. BTCS increase oxygen levels in brain tissue after impact injury(focal or diffuse injury). Examples of impact injury includecar/motorcycle accidents and falls. BTCS also augment the amount ofoxygen reaching normal brain tissue when hyper-oxygen therapy is used.

Alzheimer's Disease

BTCS increase brain oxygen consumption levels in Alzheimer's Disease,thus alleviating symptoms of Alzheimer's Disease. Blood flow and oxygenconsumption decline to level some 30% below that seen in non-dementedelderly people Wurtman, Scientific American, Volume 252, 1985.

The increased oxygen consumption levels in the brain created by BTCSalso reduce memory loss.

Diabetes

BTCS are useful for treating complications of diabetes such as ulcers,gangrene and diabetic retinopathy. Diabetic foot ulcers heal better withhyperbaric oxygen breathing treatment, M. Kalani et al. Journal ofDiabetes & Its Complications, Vol 16, No. 2, 153-158, 2002.

BTCS also help the complication of diabetic retinopathy which is relatedto low oxygen tension, Denninghoff et al., Diabetes Technology &Therapeutics, Vol. 2, No. 1, 111-113, 2000.

Other Uses

Bipolar trans carotenoid salts can also be used for the treatment ofspinal cord injury, cerebral edema, and skin papillomas. In all cases,they alleviate the condition, making it less severe. It is believed thatthis results from the increase in oxygen consumption that results fromthe use of bipolar trans carotenoid salts.

BTCS also scavenge oxygen-derived free radicals.

The following Examples are illustrative, but not limiting of thecompositions and methods of the present invention. Other suitablemodifications and adaptations of a variety of conditions and parametersnormally encountered which are obvious to those skilled in the art arewithin the spirit and scope of this invention.

EXAMPLES Example 1

Synthesis of Trans Sodium Crocetinate

Trans sodium crocetinate is synthesized by coupling a symmetrical C₁₀dialdehyde containing conjugated carbon-carbon double bonds with[3-carbomethoxy-2-buten-1-ylidene]triphenylphosphorane. This product isthen saponified using a solution of NaOH/methanol.

To ethyl bromoacetate, trephenylphosphine dissolved in ethyl acetate (ata concentration of around 2 moles/liter) is slowly added. Afterisolation, and treatment with base, the product can be treated withmethyl iodide, followed by caustic, to form the phosphorane. The basiccompound to form the carotenoid skeleton can be made starting with aring compound such as furan in this case. Furan is reacted with bromineand methanol, followed by a selective deprotonation step to form amonoaldehyde. This is then coupled with the phosphorane. Acidicconditions deprotected the other dimethyl acetal group to afford thefree aldehyde. This compound is then reacted again with the samephosphorane to give the diethyl diester. The ester groups are reduced toalcohols, and subsequent oxidation (such as with MnO₂) results in theC₁₀ skeleton in the dialdehyde form. This is later reacted with aphosphorane made from tiglic acid. The tiglic acid is esterified withmethanol under acidic conditions to give the methyl ester, followed by abromination step. The resulting allylic bromide isomers are formed, andcan be separated using crystallization. Subsequent treatment of thedesired bromide with sodium hydroxide results in the desiredphosphorane. This phosphorane and the C₁₀ dialdehyde are then dissolvedin a solvent such as toluene or benzene and refluxed. The resultingproduct isolated as a powder and is then saponified with a 40%NaOH/methanol mixture to form the TSC after solvent removal.

Trans-sodium crocetinate 1 (TSC) was prepared in a 17 step syntheticsequence in an overall yield of 1.5%. A total of 4.1 g of TSC wasprepared with ethyl bromoacetate, furan and tiglic acid as startingmaterials.

Trans-sodium crocetinate (TSC) was synthesized from saponification ofdimethyl crocetinate, the preparation of which was based on a totalsynthesis reported by Buchta and Andree.¹ The synthetic strategy behindpreparing dimethyl crocetinate was based on coupling the symmetrical C₁₀dialdehyde (2,7-dimethylocta-2,4,6-triene-1,8-dial) with(3-carbomethoxy-2-buten-1-ylidene)triphenylphosphorane.

Although the original Buchta and Andree article¹ was titled “The TotalSynthesis of trans-2,2-Bisdimethyl-crocetin-dimethyl ester andtrans-Crocetin-dimethyl ester,” experimental details and yields were notreported. Procedures for the various steps leading to the C₁₀ dialdehydeand phosphorane were found after an extensive survey of the literature.Ultimately, TSC was prepared in a 17 step sequence with ethylbromoacetate, furan and tiglic acid as the starting materials in anoverall yield of 1.5%.

The C₁₀ symmetrical dialdehyde was prepared from ethyl bromoacetate² andfuran³ using Wittig chemistry. Ethyl bromoacetate was treated withtriphenylphosphine and methyl iodide to give the phosphorane 6:

The yield for the first step was a respectable 92%. Quantitation of thesubsequent steps of this sequence were complicated by the nature ofphosphorane 4 and phosphonium salt 5. Both of these compounds wereextremely viscous syrups which foamed vigorously while concentrating ona rotary evaporator. Both compounds could be conveniently handled asmethylene chloride solutions and the overall yield of phosphorane 6appeared to be acceptable from a qualitative point of view (estimated atbetter than 75%).

Furan was ring-opened with bromine to afford fumaraldehydebis(dimethylacetal) 8.³

Mono-deprotection of bis(dimethylacetal) 8 under acidic conditions⁴ gavealdehyde 9, which was then coupled with phosphorane 6 to give 10 in a45% yield. Acidic conditions were used to deprotect the dimethylacetal10. Treating 11 with phosphorane 6 gave diester 12. The ester groupswere reduced to alcohols by DIBAL-H and subsequent oxidation with MnO₂gave the C₁₀ dialdehyde 14. The trans stereochemistry of 14 wasdetermined by NMR data. In particular, the C₂ symmetry of the compoundgave the expected 5 resonances in the ¹³C NMR spectrum and the ¹H NMRspectrum showed signals at δ9.54 (1H), 7.07 (2H) and 1.95 (3H).

The range in yields of steps h-k reflect improvements in isolation frominitial pilot studies to scaled up reactions.

Tiglic acid 15 was converted to phosphorane 20 in a 4 step sequence.Fisher esterification conditions on 15 gave the methyl ester 16.Reaction with NBS gave a mixture of 59% methyl γ-bromotiglate, 26%methyl α-bromotiglate and the balance of the material was unreactedstarting material. The formation of regioisomers was expected based onthe reported literature.⁵ In the following step, the α/γ mixture ofphosphonium salts was recrystallized to give the desired γ-phosphoniumbromide 19.⁶ Subsequent treatment with sodium hydroxide gave thephosphorane 20.

Phosphorane 20 and C₁₀ dialdehyde 14 were coupled by refluxing inbenzene.⁶ Dimethyl crocetinate 21 was isolated as a red powder.Saponification of the methyl ester proved to be more difficult thanexpected. Treating the ester 21 with 2 eq. NaOH in THF/H₂O at r.t. andreflux left the material unchanged. Solubility appeared to be asignificant problem, so pyridine was added. While this did dissolve mostof the solids, refluxing a mixture of pyridine and 2.5 N NaOH yielded noproduct. Standard THF/2.5 N NaOH saponification conditions also had noeffect on the ester. Eventually, 40% NaOH/methanol at reflux for anovernight period proved to be successful. This gave TSC 1 as an orangesolid.

Attempts were made to dissolve TSC in order to obtain a ¹H NMR spectrum.However, TSC was practically insoluble in most common organic solvents(chloroform, DMSO, pyridine, methanol, acetone, and glacial aceticacid). The TSC produced from this project was characterized by IR, UV,HPLC and elemental analyses. IR showed characteristic absorbance at 1544and 1402 cm⁻¹ (consistent with conjugated carboxylates). UV and HPLCwere consistent with authentic TSC.⁷ Elemental analyses gavesatisfactory values.

The overall yield of the reaction sequence was 1.5% (based on furan).

The synthesis is described in detail below:

All reagents and chemicals were purchased from Aldrich or Sigma and usedas received unless stated otherwise. Solvents were purchased from FisherScientific as ACS reagent or HPLC grade and used without furtherpurification. Anhydrous solvents were purchased from Aldrich inSure/Seal™ bottles and used directly without further purification.Deionized water was obtained from an in-house Culligan water treatmentsystem.

Melting points were obtained on a Mel-Temp II and were uncorrected.Infrared spectra were measured on a Perkin-Elmer 1600 FTIRspectrophotometer. Nuclear magnetic spectra were measured on a JEOLFX90Q spectrometer using a 5 mm multinuclei probe with internal orexternal deuterium lock depending on the nature of the sample. Protonand carbon NMR chemical shifts were assigned relative to TMS or thedeuterated solvent respectively. Phosphorus NMR spectra were generallyrun in the proton-decoupled mode with a coaxial insert tube of 5%aqueous phosphoric acid as the external standard.

Routine analyses by gas chromatography to evaluate reaction progress orestimate product composition were conducted on a Varian 3700 gaschromatograph equipped with a flame ionization detector and a HewlettPackard 3394A integrator. A 1 microliter solution was injected onto a 15meter DB5 column (0.53 mm D and 1.5 micron film thickness) with heliumcarrier gas using a temperature program from 50 to 250° C. at 20° C./minwith a 10 minute hold at 250° C. The injector and detector temperatureswere typically set at 250° C.

Thin layer chromatography was conducted on Baker-flex 2.5×7.5 cm silicagel plates with or without fluorescent indicator (1B2 or 1B2-F)depending on the method of detection. The components on the developedplates were detected by UV.

Elemental analyses were conducted by Quantitative Technologies, Inc.,Whitehouse, N.J.

[(Ethoxycarbonyl)methylene]triphenylphosphorane (4)² (ACL-G29-1)

Triphenyl phosphine (235.6 g, 0.90 mol) was dissolved in EtOAc (540 mL).Approximately 30 min was required for all of the solids to dissolve. Theprocess was endothermic (solution cooled to 13° C. when the ambienttemperature was 20° C.). A solution of ethyl bromoacetate (100 mL, 0.90mol) in EtOAc (400 mL) was added dropwise over a 1.5 h period. A whiteprecipitate formed during the addition. Stirred overnight (20 h) atambient temperature (18° C.).

The solids were collected by vacuum filtration rinsing with copiousamounts of Et₂O. Dried overnight in vacuo at 45° C. to give 3 as a whitesolid 356.3 g, 92.6% yield (0.83 mol). ¹H NMR was consistent withliterature values.

The solid was dissolved in methylene chloride (3 L) and treated with 1 MNaOH (3.6 L) in a 12 L flask with vigorous stirring for 45 min. Theorganic layer was separated and the aqueous phase was extracted withadditional methylene chloride (2×1 L). Organic layers were dried (MgSO₄)and concentrated until approximately 1 L of volume remained. A smallamount of material was removed and examined by ¹H NMR and found to beconsistent with literature values.

[1-(Ethoxycarbonyl)ethylidene]triphenylphosphoniun iodide (5)²(ACL-G29-2)

The material from ACL-G29-1 was treated with iodomethane (64.0 mL, 1.03mol) as the reaction flask was cooled in an ice bath. The reactionmixture was checked by TLC (silica gel, 10% MeOH/CHCl₃) when theaddition was completed (1 h) and it showed a considerable amount ofstarting material remained. The ice bath was removed and the reactionmixture was checked by TLC after 1.5 h, it looked complete based on atightening of the main band (s.m. streaked). The reaction mixture wasconcentrated on a rotary evaporator, when most of the solvent wasremoved, the product began foaming and creped up the vapor duct. Thephosphonium salt 5 appeared was an extremely viscous syrup which waskept as a methylene chloride solution to facilitate handling. Because ofthe nature of 5, the material was not quantitated.

[1-(Ethoxycarbonyl)ethylidene]triphenylphosphorane (6)² (ACL-G29-2A)

A portion of 5 dissolved in CH₂Cl₂ (350 mL) and vigorously stirred with1 M NaOH (500 mL) for 45 min. The organic layer was separated and theaqueous was extracted with CH₂Cl₂ (2×100 mL). Combined organic layerswere dried (MgSO₄) and concentrated to give 6 as a yellow solid, 8.0 g.¹H NMR spectrum was consistent with literature values.

Fumaraldehyde bis(dimethylacetal) (8)³ (ACL-G29-3)

A solution of furan (88.0 g, 1.29 mol) in anhydrous MeOH (650 mL) wascooled to −45° C. under N₂. A solution of bromine (68.0 mL, 1.32 mol)was added dropwise over a 2.5 h period at a rate to maintain ≦−45° C.The red solution was allowed to warm to −10° C. over a 2.5 h period andheld for an additional 2 h. The reaction mixture was a pale amber color.Addition of 5 g Na₂CO₃ produced a considerable amount of outgassing anda 4° C. exotherm. The reaction mixture was cooled with dry-ice and theremaining Na₂CO₃ (210 g total) was added over a 50 min period. Afterholding at −10° C. overnight (11 h, the cooling bath was removed and thereaction mixture was allowed to warm to room temperature and stirred for20 h.

The salts were removed by vacuum filtration and the filtrate was vacuumdistilled with a vigreux column until approximately 150 mL had beenremoved. Additional salt had precipitated out and was causing thedistillation pot to bump violently. After filtration, another 150 mL wasdistilled and more salt came out of solution. Once again, severe bumpingwas a problem. The still pot was cooled, filtered, the filtrate treatedwith Et₂O (400 mL) and the precipitate removed by vacuum filtration. Atleast 120 g of salt was collected (early crops of salt were discardedwithout quantitation). The majority of the Et₂O was removed on a rotaryevaporator at 25° C. with a water aspirator. Distillation was resumedwith a vigreux column, 8 was collected as a clear, colorless liquid175.2 g (76.9% yield), b.p. 86-92° C./9 torr (lit. 85-90 C/15 torr). ¹HNMR spectrum was consistent for the desired product. GC analysis: 81.9%pure.

Fumaraldyhyde mono(dimethylacetal) (9)⁴ (ACL-G29-4)

Fumaraldyhyde bis(dimethylacetal) 8 (5.29 g, 0.03 mol) was dissolved inacetone (120 mL). H₂O (1.80 mL) and Amberlyst 15 (1.20 g) weresequentially added. The mixture was stirred vigorously for 5 min thenfiltered to removed the resin. During this time, the solution turnedfrom colorless to yellow. The filtrate was concentrated on a rotaryevaporator at room temperature and the light brown residue was distilledon a kugelrohr (37° C./200 millitorr) to give 9 as a yellow liquid, 2.80g, 71.8% yield. A small amount of material was lost when the still potbumped at the beginning. ¹H NMR spectrum was consistent for the desiredproduct, GC analysis indicated 80% purity.

(ACL-G29-7)

Fumaraldyhyde bis(dimethylacetal) δ 72.1 g, 0.41 mol) was dissolved inacetone (1600 mL). H₂O (25.0 mL) and Amberlyst 15 (16.7 g, prewashedwith acetone) was added. The mixture was stirred vigorously for 5 minthen filtered to removed the acid resin. The reaction mixture had aslight yellow tint, much fainter than the previous large scale prep. GCanalysis indicated 34.5% product and 46.1% s.m. Treated with resin foranother 5 min. GC analysis indicated 59.5% product and 21.7% s.m.Treated with resin for another 10 min (total time 20 min). GC analysisindicated 73.9% product and 2.0% s.m. The filtrate was concentrated on arotary evaporator at room temperature to give a brown oil, 54 g. Vacuumdistillation gave a yellow-green oil, 34.48 g. GC analysis indicated64.7% purity (8.22 min) with a major impurity of 17.5% (9.00 min) and6.9% (9.14 min). Net recovered yield 22.3 g (0.17 mol). Analysis of theforecut by GC showed extremely dirty material.

(ACL-G29-13)

Amberlyst 15 (8.61 g) was stirred in acetone (100 mL) for 30 min andcollected by filtration. The acetal 8 (35.0 g, 0.16 mol) was dissolvedin acetonitrile (620 mL) and while mechanically stirred, acid resin anddeionized H₂O (10.0 mL, 0.55 mol) was added. The course of the reactionwas monitored by TLC (10:3 hexane:Et₂O), after 15 min most of thestarting material had been converted. After 20 min, only a trace of thedimethyl acetal was detected. The resin was removed by filtration andthe filtrate was concentrated on a rotary evaporator at ≦40° C. Thecrude product was loaded on a Biotage column (7.5×9.0 cm) eluting with15% Et₂O in hexanes to give 19.8 g. 65% yield.

6,6-Dimethyoxy-2-methylhexa-2,4-dienoate (10)² (ACL-G29-5)

The ylide 6 (7.80 g, 22 mmol) was dissolved in methylene chloride (65mL). A solution of fumaraldehyde mono(dimethylacetal) 9 (2.80 g, 17mmol) was added and the solution was stirred overnight. Solvent wasremoved at reduced pressure on a rotary evaporator. ¹H NMR of the crudeindicated desired product was present. Upon standing, crystals grew(presumably triphenylphosphine oxide). The solid (14.1 g after drying byvacuum filtration) was slurried in petroleum ether and filtered. Thefiltrate was concentrated to give a yellow oil with solids precipitatedout which was dissolve in methylene chloride (15 mL) and chromatographedon a Biotage 4×7.5 cm column eluting with methylene chloride to give 10as a yellow oil 1.8 g, 50% yield. ¹H NMR spectrum of the yellow oil wasconsistent literature values, however, a trace of methylene chlorideremained (0.75 eq) so the material was place on the rotary evaporatorfor 45 min. Mass was reduced to 1.5 g, 40.6% yield and the methylenechloride resonance disappeared. GC analysis major peak at 12.6 min,87.5% (50° C., 5 min hold, 20° C./min to 250° C. final temperature).

(ACL-G29-6)

A solution of ylide 6 (59.2 g, 0.16 mol) in methylene chloride (650 mL)was cooled in an ice bath and a solution of 9 (25.7 g, 0.19 mol) wasadded. The solution was stirred overnight allowing the ice bath to melt.TLC (hexane:Et₂O 10:3) indicated at least 3 other compounds running veryclose to the product. Examination of the aldehyde indicated by GCanalysis 50.0% purity. Solvent was removed to give a solid/oil mixture.

(ACL-G29-8)

Ylide 6 (59.2 g, 0.16 mol) and acetal 9 (0.19 mol) was coupled inmethylene chloride (1.1 L) and worked up as described above to give ayellow-green oil, 80 g. A portion of the crude reaction mixture (4.13 gof the original 80 g) was placed on the kugelrohr and distilled at 50°C./250 millitorr. A colorless oil was condensed 2.28 g, ¹H NMR indicatedit was the starting aldehyde while the product 10 remained in the stillpot, 1.85 g. Volatile components were removed from the bulk of the crudeproduct by kugelrohr distillation at 50° C./200 millitorr (net 35 g).

Ethyl 2-methyl-6-oxo-hexa-2,4-dienoate (11)² (ACL-G29-9)

Acetal 10 from the pilot still pot (ACL-G29-8, 1.85 g, 9 mmol) wasdissolved in acetone (33 mL). Deionized H₂O (0.50 mL) and Amberlyst 15resin (0.35 g, prewashed with acetone) were added. The mixture wasstirred for 20 min. Filtered and concentrated on a rotary evaporator togive a yellow-green oil, 1.53 g. Chromatographed on a 4.5×7 cm Biotagecolumn eluting with 15% Et₂O in hexanes. This system gave incompleteseparation, but 0.32 g of the main component was isolated and analyzed;¹H NMR spectrum was consistent with literature data and IR (1711, 1682cm⁻¹) was consistent with the desired product. GC 95.6%. An additional0.35 g was recovered, although it was cross contaminated with less andmore polar material. The ¹H NMR spectrum indicated fairly cleanmaterial. GC 90.6%. Yield: 42%.

Diethyl 2,7-dimethylocta-2,4,6-triene-1,8-dioate (12)² (ACL-G29-10)

The aldehyde 11 (0.65 g, 3.5 mmol) from G29-9 was dissolved andmagnetically stirred in methylene chloride. Ylide (1.59 g, 4.4 mmol) wasadded. The light yellow-green solution turned a darker shade yellowwithin minutes. TLC after 10 min indicated starting material was almostcompletely consumed. After stirring for 20 h, the reaction mixture(brown solution) was filtered through a pipette partially filled withsilica gel. The filtrate was concentrated to give a brown solid. Thesolid was dissolved in 5% Et₂O in hexanes with a small amount of CHCl₃.Chromatographed on a 4×7.5 cm Biotage column eluting with 5% Et₂O inhexanes. The main product was isolated as a white crystalline solid, 045g, 50% yield. ¹H NMR spectrum was consistent with literature data.

(ACL-G29-14)

An additional amount of 12 was prepared as described above to give 21.8g, 81.6% after chromatographic purification. ¹H NMR spectrum wasconsistent with the desired product.

2,7-Dimethylocta-2,4,6-triene-1,8-diol (13)² (ACL-G29-11)

The diester 12 (0.45 g, 1.8 mmol) was taken up in anhydrous hexanes(15.0 mL). It appeared as though some of the material dissolved, but themixture was quite cloudy. More material appeared to come out of solutionwhen the mixture was cooled in a −78 C bath. Neat DIBAL-H (2.50 mL) wasdissolved in anhydrous hexanes (total volume 10.0 mL) and a portion(approximately 2 mL) was inadvertently siphoned into the reactionmixture as the diester was cooled in a dry-ice bath. An additionalamount of DIBAL-H solution was added until a total of 5.0 mL (6.7 mmol)was added. The CO₂ bath was allowed to warm. After stirring for 2 h 50min, TLC indicated the diester was completely consumed. Bath temperaturewas adjusted to −20° C. allowing to warm to 0° C. over 20 min. Treatedwith H₂O/silica gel (2 mL/7 g) mixture for 30 min. Added K₂CO₃ andMgSO₄. Filtered to remove the solids and thoroughly rinsed withmethylene chloride. Concentrated to give a white solid, 0.14 g, 50%yield. Note: TLC R_(f)=0.21 (5% MeOH/CHCl₃) is quite polar. Rinsing withmethylene chloride might not have been enough to recover all of theproduct. ¹H NMR spectrum was consistent with literature values.

(ACL-G29-15)

The diester (5.4 g, 21 mmol) was taken up in anhydrous hexanes (175 mL,poor solubility), cooled in a −78° C. bath and treated with a solutionof DIBAL-H (14.5 mL in 50 mL anhydrous hexanes) over a 35 min period.Vigorous gas evolution was observed during the addition. The color ofthe slurry went from white to dark yellow initially, this lightened upas additional DIBAL-H was added. Allowed to warm to −40° C. over 2 h,then transferred to a −28° C. bath overnight. The reaction mixture wastreated with a homogeneous mixture of H₂O/silica gel (4 mL/14.4 g) for30 min. MgSO₄ (7.5 g) and K₂CO₃ (5.1 g) was added and the reactionmixture was removed from the cooling bath. Stirred 20 min, then filteredon a sintered glass funnel. The solids were washed with methylenechloride—this caused a considerable amount of precipitate to form.Warming while placed on a rotary evaporator dissolved the precipitatedsolids. The solids remaining on the sintered glass funnel was washedwith EtOAc (4×75 mL) and the filtrate was concentrated.

CH₂Cl₂ rinsings gave a pale-yellow solid, 1.7 g, ¹H NMR was consistentwith literature values; EtOAc rinsings gave an off-white solid, 1.0 g,¹H NMR consistent with literature values; total recover 2.7 g, 75%yield.

(ACL-G29-17)

The diester (16.4 g, 6.5 mmol) was stirred in anhydrous hexanes (500 mL)under N₂ and cooled to −78° C. A solution of DIBAL-H (45 mL, 253 mmol)in hexanes (150 mL) was added over a 1 h period. Allowed to warm to −30°C. and stirred overnight (17.5 h total time). A homogeneous mixture ofH₂O/silica gel (12.3 g/43.7 g) was added and the mixture was manuallyswirled over a 45 min period. Added K₂CO₃ (15.5 g) and MgSO₄ (23.5 g).Swirled over another 30 min period. Filtered on a sintered glass funnel,rinsed with methylene chloride (ppt formed, presumably caused byevaporative cooling) and the filtrate was concentrated. The solids wererinsed with several times with EtOAc (approximately 100 mL portions, 2 Ltotal volume) and pooled with the original filtrate. Concentrated togive a yellow solid, 8.9 g, 81% crude yield. ¹H NMR spectrum wasconsistent with the desired product.

2,7-Dimethylocta-2,4,6-triene-1,8-dial (14)² (ACL-G29-12)

A slurry of MnO₂ (7.80 g, 90 mmol) was cooled in an ice bath under N₂. Asolution of diol 13 (0.14 g, 0.8 mmol) was added via pipette as anacetone solution (5.0 mL). An additional 2.0 mL of acetone was used torinse the flask and complete the transfer. The ice bath was allowed tomelt overnight as the reaction mixture was stirred. Solids were removedby filtration through Hyflo and concentrated to give a yellow solid. Thematerial was dissolved in 10% Et₂O/hexanes with a minimal amount ofCHCl₃ and applied to a column of silica gel (30×190 mm) eluting with 10%Et₂O/hexanes. The product could be followed as a yellow band as iteluted, 14 was isolated as a light yellow solid 37 mg, 26% yield. ¹H NMRspectrum was consistent literature values.

(ACL-G29-16)

A solution of the diol 13 (2.70 g, 16 mmol) in acetone (500 mL) wascooled in an ice bath under N₂. MnO₂ (60.0 g, 0.69 mol) was added inportions over a 20 min period. The ice bath was allowed to melt as thereaction mixture was stirred overnight. The reaction mixture wasfiltered through Hyflo and the filtrate was concentrated to give ayellow solid, 1.6 g, 61% crude yield. ¹H NMR was consistent with theliterature values. The crude yellow solid was dissolved in methylenechloride (along with a small amount of 10% Et₂O in hexanes was added)and charged to a 4×7.5 cm Biotage silica gel column. Eluted initiallywith 10% ether in hexanes (1 L), then increased polarity to 15% Et₂O (1L) and 20% Et₂O (0.5 L). Recovered a yellow solid 1.0 g, 38% yield. ¹HNMR spectrum consistent with desired product.

(ACL-G29-21)

A solution of the diol (9.31 g, 60 mmol) in acetone (500 mL) was cooledin an ice bath under N₂. MnO₂ (100 g, 1.15 mol) was added and themixture was stirred as the ice bath was allowed to melt overnight.Checked by IR after 24 h, significant amount of product had formed, butstill quite a bit of alcohol present. Added an additional 50 g ofoxidant and continued stirring for another overnight period. A portionof the reaction mixture was filtered and checked by ¹H NMR, the reactionappeared complete based on the consumption of starting material. Therest of the reaction mixture was filtered through a pad of Hyflo andthoroughly rinsed with acetone. Concentrated to give a dark yellowsolid. Azeotroped once with 40 mL benzene then dried in vacuo at 40° C.for 5 h, then at r.t. overnight. Recovered 5.28 g, 58% yield. ¹H NMR andIR spectra were consistent for the desired product.

Methyl Tiglate (16)

In a 2 L 3-neck flask fitted with an overhead stirrer, condenser andthermometer, a solution of tiglic acid 15 (89.8 g; 0.9 mol) and 5 mLconcentrated sulfuric acid (0.09 mol) in 900 mL methanol was heated atreflux for 20 hrs. The solution was cooled to 25° C. and the excessmethanol was stripped at 30° C. and 27 in Hg vacuum on a rotaryevaporator. GLC analysis of the recovered methanol distillate showedproduct in the overheads. The resulting two-phase, light brownconcentrate was taken up in 500 ml ethyl ether and washed successivelywith 250 mL water, 250 mL 10% aqueous sodium bicarbonate and 250 mLsaturated brine. The ether solution was dried over anhydrous potassiumcarbonate, filtered and stripped on the rotary evaporator at 25° C. and17 in Hg vacuum to give crude methyl tiglate as a near colorless oil;43.6 g (42% yield). GLC analysis showed one major volatile product witha retention time of 2.7 min compared to 3.8 min for the starting tiglicacid. Proton NMR in CDCl₃ showed the expected signals with some traceethyl ether contamination: 1.79 ppm (d, 3H), 1.83 (s, 3H), 3.73 (s, 3H),6.86 (q, 6.6 Hz). IR (neat on KBr): ester carbonyl at 1718 cm⁻¹. Thisoil was used as is in the next step.

Methyl γ-Bromotiglate (17)⁵

In a 1 L 4-neck flask fitted with an overhead stirrer, a thermometer anda condenser, a stirred mixture of the crude methyl tiglate (43.6 g; 0.38mol), N-bromosuccinimide (68 g; 0.38 mol) and 70% benzoyl peroxide (5.34g; 0.015 mol) in 500 mL carbon tetrachloride was heated at reflux fortwo hours. After cooling to 20° C., the insoluble succinimide (38.1 g100% recovery) was suction filtered off. The filtrate was washed threetimes with 250 mL water, dried over MgSO₄ and then stripped on a rotaryevaporator at 25° C. and 26 in Hg vacuum to give a yellow oil; 78.8 g.Proton NMR of this oil in CDCl₃ gave a complex spectrum. The methyleneprotons for the desired γ-bromo ester were assigned to the doubletcentered at 4.04 ppm (8.6 Hz), while the same protons for the α-bromoisomer were ascribed to the singlet at 4.24 ppm. Proton integration ofthese signals and the methyl multiplet from 1.6 to 2.0 ppm suggested thefollowing composition (mole %):

-   -   γ-bromo ester: 59%    -   α-bromo ester: 26%    -   starting material: 15%

This crude oil was used in the next step without any furtherpurification.

This reaction was also run on a 0.05 mole scale using only 0.87equivalents of N-bromosuccinimide under otherwise identical conditions.The composition of this crude oil was estimated based on its proton NMRspectrum as 52% γ-bromo ester, 24% α-bromo ester and 23% unreactedmethyl tiglate. GLC analysis of this oil was slightly more complicatedshowing other minor components.

Triphenylphosphonium Salt of Methyl γ-Bromotiglate (19)⁶

In a 2 L 4-neck flask fitted with a thermometer, a 100 mL constantpressure addition funnel and a condenser connected to a static nitrogensystem, a stirred solution of the crude methyl γ-bromotiglate (78.8 g)in 350 ml benzene was treated dropwise with a solution oftriphenylphosphine (95 g; 0.36 mol) in 350 mL benzene over a period of1.75 hrs. The temperature of the mixture exothermed slightly from 24 to27° C. under otherwise ambient conditions. After the addition, thereaction was stirred vigorously overnight to afford a slurry of whitesolid containing a yellowish gum that adhered to the walls of the flask.The white solid was suction filtered onto a sintered glass funnelwithout disturbing the yellowish gum. The flask was washed twice with100 mL benzene and poured onto the filter. The filter cake was washedwith 50 mL benzene and then twice with 50 mL hexane. The wet cake wasdried in a vacuum oven at ambient temperature for 5.5 hours. The driedwhite powder [93 g; mp=125° C. dec)] was dissolved in 150 mLacetonitrile with heat to give a clear yellow solution. Ethyl acetate(300 mL) was added to this hot solution and the product started tocrystallized after adding about 100 mL ethyl acetate. The flask wasstored in the refrigerator overnight. The product was suction filteredand washed with a minimum amount of 1:2 acetonitrile and ethyl acetate;45.0 g. mp=187-190° C. (dec). lit mp=183° C. (dec).

The gummy solids in the reaction flask were recrystallized from 10 mLacetonitrile and 20 mL ethyl acetate. Also, additional solidsprecipitated overnight from the benzene mother liquor. These solids werefiltered and recrystallized in the same manner. Both samples wererefrigerated for 2 hours and suction filtered to give additionalproduct; 13.3 g.

The benzene filtrate was stripped on a rotary evaporator and the yellowoil taken up in 10 mL acetonitrile and precipitated with 20 mL ethylacetate. The slurry was stored in the refrigerator overnight to giveadditional product as a white solid; 4.6 g. m.p. 185-187° C. (dec).Total yield of the desired phosphonium salt as a white solid was 62.9 gor 36.2% yield based on the crude methyl tiglate. Proton NMR (CDCl₃,TMS) ppm 1.55 (d, 4 Hz, 3H), 3.57 (s, 3H), 4.9 (dd, 15.8 & 7.9 Hz, 2H),6.55 (broad q, 6.6-7.9 Hz, 1H), 7.4-7.9 (m, 15H). Proton-decoupledPhosphorus NMR (CDCl₃, 5% aq H₃PO₄ coaxial external standard) 22.08 ppm.Partial Carbon NMR (CDCl₃): CO₂CH₃, (166.6 ppm, d, J_(CP)=3 Hz),olefinic CH (117.5 ppm, d, J_(CP)=86.1 Hz), CO₂ CH₃, (52.0 ppm),Ph₃P—CH₂ (25.4 ppm, d, J_(CP)=50.6 Hz) and CH₃ (13.4 ppm, d, J_(CP)=2.4Hz). Partial IR (KBr pellet): ester carbonyl at 1711 cm⁻¹.

(3-Carbomethoxy-2-buten-1-ylidene)triphenylphosphorane (20)⁶

In a 5 L 5-neck flask fitted with an overhead stirrer, an additionfunnel and a thermometer, a solution of sodium hydroxide (5.12 g; 0.128mol) in 250 ml water was added dropwise to a vigorously stirred solutionof the triphenylphosphonium salt of methyl γ-bromotiglate (58.3 g; 0.128mol) in 2,500 mL water over a period of 41 minutes at 25° C. The yellowslurry was stirred for 10 minutes at room temperature and then suctionfiltered. The filter cake was washed with 1,800 mL water and thenthoroughly dried on the filter with a nitrogen blanket. The yellow solidwas then dried overnight in a vacuum desiccator over P₂O₅ at roomtemperature and 27″ Hg vacuum; 35.3 g (73.7% yield). mp=145-150° C. litmp=145-165° C. Proton-decoupled phosphorus NMR in CDCl₃ showed two peaksat 17.1 ppm and 21.1 ppm in a ratio of 93:7. Proton NMR (CDCl₃, TMS) ppm1.89 (s, 3H), 3.58 (s, 3H), 7.3-7.8 (m, 17H). A small but detectablesinglet at 1.74 ppm was also apparent in this spectrum which wasattributed to the impurity. This solid was used without furtherpurification in the next step.

Dimethyl crocetinate (21)⁶ (ACL-G29-18)

The dialdehyde 14 (0.48 g, 2.9 mmol) was added to a 100 mL round bottomflask. Benzene (20 mL) was added and the solids were dissolved withmagnetic stirring. The ylide was added, an additional 10 mL benzene wasused to wash the compound into the flask. Warmed to a vigorous refluxfor 6 h. The reaction mixture was allowed to cool overnight. Contrary toliterature reports, a very small amount of solid had formed. Thereaction mixture was concentrated, the residue was taken up in MeOH (30mL) and boiled for 30 min. Upon cooling to ambient temperature, thesolids were collected by vacuum filtration. An NMR sample was preparedby dissolving 20 mg into 0.5 mL CDCl₃, somewhat surprisingly, thisrequired warming with a heatgun to dissolve completely. ¹H NMR spectrumwas recorded and found to be consistent with the desired product. Theremaining material was dissolved in hot benzene, filtered, the filtratewas concentrated, taken up in MeOH, cooled in an ice bath and solids redsolids were collected, 334 mg, 33% yield. This material did not appearto be any more soluble than the material which was originally isolated.

(ACL-G29-18A)

Dialdehyde 14 (5.78 g, 35 mmol) was dissolved in benzene (300 mL) underN₂. Ylide 20 (35.3 g, 94 mmol) was added and the mixture was warmed toreflux for 6 h forming a dark red solution. After allowing the reactionmixture to cool overnight, red solids were collected by vacuumfiltration and rinsed with methanol. Transferred to a 500 mL RBF andrefluxed with approximately 65 mL methanol for 30 min. Cooled andcollected a red solid. Rinsed with cold methanol and dried in vacuo togive 21 as a red solid, 3.00 g. ¹H NMR and IR spectra were consistentwith the desired product.

The original filtrate (from the reaction mixture) was concentrated on arotary evaporator and the dark residue was taken up in 100 mL methanoland refluxed for 40 min. Cooled in an ice bath and collected by vacuumfiltration a red solid. Rinsed with cold methanol and dried in vacuo togive 21 as a red solid, 1.31 g. ¹H NMR spectrum was consistent with thedesired product.

The filtrates were pooled, concentrated and taken up in 75 mL methanoland allowed to sit overnight at r.t. A red solid was recovered by vacuumfiltration: 0.38 g. ¹H NMR spectrum was consistent with the desiredproduct.

More solids had formed in the filtrate. Isolated by vacuum filtration togive a red solid, 0.127 g. IR consistent with above. Total recovery:4.89 g, 39% yield.

Saponification Attempt with THF/NaOH (ACL-G29-19)

A stirred suspension of diester 21 (100 mg, 0.28 mmol) in THF (2 mL) and1N NaOH (0.56 mL, 2 eq) was added. Stirred at r.t. overnight. TLC showedonly starting material. Warmed to reflux, no change after several hours.Added THF (6 mL) in an attempt to dissolve more of the solids, but itdidn't seem to matter. Continued refluxing overnight. Added more THF(about 6 mL, TLC showed only starting material), and refluxed foranother overnight period. Concentrated and check by ¹H NMR—only startingmaterial (based on integration of the methyls and methyl esters).Dissolved in pyridine (to mL) while warmed on a heating mantle. Added2.5 N NaOH (1.0 mL). The dark orange solution turned deep red afterseveral minutes. The heating mantle was removed, solids began forming,mantle reapplied for 30 min, then stirred at r.t. overnight.Concentrated on high vacuum. The residue was insoluble in chloroform,DMSO, pyridine and sparingly soluble in H₂O. An IR (Nujol mull) showedC═O absorbance characteristic of the starting material.

Saponification with 2.5 N NaOH and THF (ACL-G29-20)

Diester 21 (37 mg, 0.10 mmol) was weighed into a flask and stirred indiethyl ether (4 mL). The solvent took on an orange color, but solidswere still present. Added 1 mL of 2.5 N NaOH and warmed to reflux. Afterhalf an hour, most of the ether had evaporated. This was replaced withTHF (3 mL) and refluxing was continued for several hours. Solid werecollected by vacuum filtration, rinsed with deionized water then driedin a vacuum oven. IR showed only starting material.

Saponification with 40% NaOH (1) (ACL-G29-22)

Diester 21 (32 mg, 8.9 mmol) was weighed into a flask and stirred inmethanol (1.5 mL). The solvent took on an orange/red color, but solidswere still present. Added 1.5 mL of 40% NaOH and warmed to reflux for 17h. After cooling to r.t., orange solids were collected by vacuumfiltration and rinsed with deionized water. Dried in vacuo at 40° C. togive 1 as an orange powder 21 mg, 59%. IR (KBr pellet) 3412, 1544, 1402cm⁻¹, the compound is probably hygroscopic, upfield carbonyl shift isconsistent with conjugation.

(ACL-G29-22A)

Repeated with 35 mg of diester 1 refluxing for 15 h. The reactionmixture was cooled in an ice bath, collected by vacuum filtration andwashed with cold deionized water. Dried in vacuo at 40° C. Recovered 1as an orange solid 25.5 mg, 65%.

(ACL-G29-23)

Diester 21 (0.48 g, 1.3 mmol) was taken up in methanol (15.0 mL) and 40%sodium hydroxide (15.0 mL) and warmed to reflux. The heterogeneous redmixture turned orange after about 2 h. Heating was discontinued after 6h and the mixture was allowed to cool overnight. An orange solid wascollected by vacuum filtration and washed with cold deionized water.Drying in vacuo gave a friable orange solid, 0.36 g, 68% yield.

(ACL-G29-24)

Diester 21 (1.10 g, 3.1 mmol) was placed in a 100 mL recovery flask andheated to reflux in methanol (20 mL) and 40% NaOH (20 mL) for 12 h.After cooling in an ice bath, an orange solid was collected by vacuumfiltration and rinsed with deionized water. Drying in vacuo gave 1.4 g,100%. Anal Calcd for C₂₀H₂₂O₄Na₂-0.4H₂O: C, 63.29; H, 6.05; Na, 12.11;H₂O, 1.90. Found: C, 63.41; H, 6.26; Na, 11.75; H₂O, 1.93.

(ACL-G29-25)

Diester 21 (3.00 g, 8.4 mmol) was refluxed in methanol (80 mL) and 40%NaOH (60 mL) for 12 h. The product was isolated as an orange solid asdescribed above 2.7 g, 80%. Anal Calcd for C₂₀H₂₂O₄Na₂-0.4H₂O: C, 63.29;H, 6.05; Na, 12.11; H₂O, 1.90. Found: C, 63.20; H, 6.00; Na, 11.93; H₂O,1.81. Samples ACL-G29-23, -24 and -25 were ground on an agate mortar andcombined as ACL-G29-A.

REFERENCES

-   1. E. Buchta and F. Andree Naturwiss. 1959, 46, 74.-   2. F. J. H. M. Jansen, M. Kwestro, D. Schmitt, J. Lugtenburg Recl.    Trav. Chim. Pays-Bas 1994, 113, 552.-   3. R. Gree, H. Tourbah, R. Carrie Tetrahedron Letters 1986, 27,    4983.-   4. G. M. Coppola Syn. Commun. 1984, 1021.-   5. D. S. Letham and H. Young Phytochemistry 1971, 10, 2077.-   6. E. Buchta and F. Andree Chem. Ber. 1960, 93, 1349.

Example 2

Synthesis of Trans Potassium Norbixinate

Trans potassium norbixinate is synthesized by coupling a symmetrical C20dialdehyde containing conjugated carbon-carbon double bonds with[1-(ethoxycarbonyl)methylidene]triphenylphosphorane. The preparation ofthis compound is similar to that listed previously for trans sodiumcrocetinate, except that the furan starting material is replaced withthe appropriate ringed structure. This product is then saponified usinga solution of KOH/methanol.

Example 3

Synthesis of a Longer BTCS

The above compound is synthesized by adding a symmetrical C₁₀ dialdehydecontaining conjugated carbon-carbon double bonds to an excess of[3-carbomethoxy-2-buten-1-ylidene]triphenylphosphorane. The preparationof this compound is similar to that listed previously for trans sodiumcrocetinate, except that the furan starting material is replaced withthe appropriate ringed structure. The trans 40-carbon product is thenisolated using a procedure such as chromatography. This product is thensaponified using a solution of NaOH/methanol.

Example 4

TSC by Inhalation

TSC has been given to rats via an inhalation route. Ten rats were givenTSC directly into the lungs. This was done by inserting a tube into thetrachea, and nebulizing 0.2 ml of TSC solution (TSC dissolved in dilutesodium carbonate solution) with about 3 to 6 mls of air. For all dosagesstudied (0.5-2 mg/kg), about 20% of the drug was present in the bloodstream within one minute after it was given. For dosages of 0.8-1.6mg/kg the drug was present in the blood stream for a period of at leasttwo hours.

Example 5

Improved Synthesis Method

Prep of Tetraethyl 2-Butenyl-1,4-bisphosphonate

A 250 mL 3-neck flask was equipped with a Teflon-coated thermocouple, a60 mL constant pressure addition funnel and a simple distillation head.Under a nitrogen atmosphere, neat triethyl phosphite (59 mL; 0.344 mol)was heated with a heating mantle controlled with a JKem controller at140° C. A solution of trans-1,4-dichloro-2-butene (26.9 g; 0.215 mol)and triethyl phosphite (35 mL; 0.204 mol) was added dropwise at 134-144°C. over a period of 93 minutes. The clear solution was then kept at 140°C. under nitrogen. After 37 minutes, gas chromatography of an aliquot (1drop) in 1 mL of ethyl acetate showed desired product, intermediateproduct and the two starting materials.

After 15.5 hrs at 140° C., gas chromatography of an aliquot (1 drop in0.5 mL EtOAc) showed the desired product with no detectable startingdichloride or intermediate product. After 16 hrs, the faint yellowsolution was cooled to room temperature under nitrogen. The faint yellowoil was distilled in a Kugelrohr with a two-bulb receiver and thefurther bulb cooled in a dry ice-acetone bath at 25-100° C. and 0.1-0.2torr to give a colorless oil (14.8 g) as a forecut. Gas chromatographyshowed only product in the Kugelrohr pot. This light amber oil wasdistilled in a Kugelrohr at 140° C. and 0.1-0.15 torr to give distillateas a colorless oil; 66.45 g (94.1% yield). Gas chromatography showedonly one volatile component. GC-MS analysis showed that this componentwas the desired product, giving a small molecular ion at 328 m/z and abase ion at 191 m/z (loss of PO₃Et₂). Proton NMR was consistent with thedesired product. Carbon NMR also was consistent with the desiredbis(phosphonate diester), showing only long range (W-coupling) andnormal carbon-phosphorus coupling to the allylic carbon.

Pot residue—light yellow oil—0.8 g.

Prep of 1,1,8,8-Tetramethyoxy-2,7-dimethyl-2,4,6-ocatriene

Under a nitrogen atmosphere, a magnetically stirred mixture oftetraethyl trans-2-butenyl-1,4-bisphosphonate (3.3 g; 10.0 mmol),pyruvic aldehyde dimethyl acetal (2.6 mL; 21.5 mmol) in 10 mL tolueneand 10 mL cyclohexane was treated successively with anhydrous potassiumcarbonate (10.2 g; 73.8 mmol) and powdered sodium hydroxide (1.25 g;31.2 mmol). The solution turned yellow immediately. The resulting slurrywas stirred at ambient temperature under nitrogen. The reaction slowlyexothermed, reaching a maximum of 38° C. after about 25 minutes. Also, agummy precipitated formed, which negatively impacted magnetic stirring.After 2.5 hrs, gas chromatography of an aliquot of the yellow-orangesolution (1 drop in 0.5 mL toluene) showed the two starting materialsand 3 other new components.

After 16.75 hrs at ambient temperature, gas chromatography of an aliquotof the orange solution (1 drop in 0.5 mL toluene) showed only a smallamount of the starting bis(phosphonate diester). The resulting orangemixture with a gummy mass (unable to stir) was cooled in an ice bath andquenched with 100 mL 10% aqueous NaCl. The solids were dissolved in thisaqueous solution by working with a spatula. The mixture was thenextracted with 200 mL 1:1 ether:hexane. The organic layer was washedwith 10% aqueous NaCl (200 mL) and then saturated brine (100 mL). Thecolorless organic layer was dried over Na₂SO₄. Gas chromatography showedthree major components and no detectable starting bis(phosphonatediester). The thin layer chromatogram showed two major spots and oneminor spot. The Na₂SO₄ was suction filtered off and washed with ether.The filtrate was concentrated on a rotary evaporator at 35° C. to give acolorless oil; 1.8 g. GC-MS Analysis showed that the three majorvolatile components were the isomeric products, giving molecular ions at256 m/z and base ions at 75 m/z [(MeO)₂CH⁺]. Proton NMR also wasconsistent with a mixture of isomeric products along with otherunidentified impurities. Yield of crude product=70.3%.

Prep of 1,1,8,8-Tetramethyoxy-2,7-dimethyl-2,4,6-ocatriene

A mechanically stirred mixture of tetraethyltrans-2-butenyl-1,4-bisphosphonate (63.2 g; 0.19 mol), pyruvic aldehydedimethyl acetal (50 mL; 0.41 mol) in 200 mL toluene and 200 mLcyclohexane was treated successively with anhydrous potassium carbonate(196 g; 1.42 mol) and powdered sodium hydroxide (24.0 g; 0.60 mol). Thesolution turned yellow immediately. The resulting slurry was stirred atambient temperature under nitrogen. The reaction exothermed to 61° C.after about 11 minutes and the stirred mixture was cooled in an ice bathto drop the temperature to 35° C. After 4.7 hrs at 29-35° C., gaschromatography of an aliquot (3 drops in 0.5 mL toluene) showed nostarting bis(phosphonate). After 5 hrs, the mixture was cooled in an icebath to 13° C. and 10% aqueous sodium chloride (400 mL) was added as thetemperature rose to 30° C. More 10% aqueous sodium chloride (1,500 mL)was added and the mixture was extracted with 3,000 mL 1:1 ether:hexane.The tinted yellow organic layer was washed with 10% aqueous sodiumchloride (2×1,000 mL) and then with saturated brine (1,000 mL). Thetinted yellow organic layer was dried over Na₂SO₄, filtered andconcentrated on a rotary evaporator at 30° C. to give a light yellowoil; 43.4 g. Gas chromatography showed three major components comprising89% of the mixture with no detectable starting bis(phosphonate). TLCanalysis showed one major and 3 minor components.

Proton NMR showed isomeric product plus toluene. The oil was evaporatedfurther on a Kugelrohr at 50° C. and 0.2 torr for 30 minutes; 31.9 g.Proton NMR showed isomeric bis(acetal) product with no detectabletoluene.

Yield=65.5%

Prep of 2,7-Dimethyl-2,4,6-ocatrienedial at Higher Payload

Under a nitrogen atmosphere, a magnetically stirred solution of crude1,1,8,8-tetramethyoxy-2,7-dimethyl-2,4,6-ocatriene isomers (31.9 g;124.4 mmol) in tetrahydrofuran (160 mL), water (80 mL) and glacialacetic acid (320 mL) was heated at 45° C. with a heating mantlecontrolled with a JKem controller via a Teflon-coated thermocouple (9:03am). After 30 minutes, the mixture exothermed to a maximum of 54° C. andthen returned to the 45° C. setpoint. Gas chromatography of an aliquot(3 drops in 0.5 mL THF) after 3 hours showed some residual startingmaterial, two major and one minor product. The yellow reaction solutionwas cooled in an ice bath to 21° C. and then diluted with 4:1ether:dichloromethane (2,000 mL). This solution was then washedsuccessively with 20% aqueous NaCl (2,000 mL×2), 4:1 20% aq NaCl: 1Maqueous NaOH (2,000 mL×3)¹ and 20% aq NaCl (1,000 mL×2). The yelloworganic layer was dried over MgSO₄, filtered and concentrated on arotary evaporator to give a yellow solid; 18.9 g. Gas chromatographyshowed one major and one minor component starting bis(acetal). TLCanalysis showed one major spot and several minor, more polar impurities.This solid was dissolved in 250 mL refluxing methanol, cooled to roomtemperature and then in an ice bath for 1 hr. The slurry was suctionfiltered to give a yellow fluffy needles; 14.15 g. Gas chromatographyshowed 95:5 mixture of isomeric dialdehydes. This solid wasrecrystallized again with 200 mL refluxing methanol, cooled to roomtemperature and then in the refrigerator overnight.¹ The first two washes apparently removed acetic acid as evident byneutral pH. The third wash turned red and was still basic, suggestingremoval of byproduct.

The slurry was suction filtered and washed with freezer-chilled methanolto give yellow needles; 11.2 g. Gas chromatography showed 97:3 mixtureof isomeric dialdehydes. TLC analysis showed one spot. The needles weredried in a vacuum oven at 45° C. for 160 minutes until constant weight;10.75 g.

uncorrected mp=154-156° C. lit² mp=161-162° C. Proton NMR and Carbon NMRwere consistent with the desired symmetrical dialdehyde.² Dictionary of Organic Compounds. Verson 10:2, September, 2002.

The two methanol filtrates from the recrystallizations were combined.The thin layer chromatogram showed product plus other impurities. Thefiltrates were concentrated and various crops collected as shown below.Crop Appearance Amt (g) Isomeric Ratio 2 yellow powder 1.4 80:20 3yellow needles 2.6 75:25 4 yellow solid 4.45 46:30

Crop 2 & 3: These combined crops were dissolved in 20 mL refluxing ethylacetate, cooled to room temperature and then in the freezer for 1 hr.The slurry was suction filtered and washed with freezer-chilled ethylacetate to give yellow needles; 1.95 g. Gas chromatography showed 86:14mixture of isomers. This solid was recrystallized again in ethyl acetate(10 mL) to give yellow needles; 1.55 g. Gas chromatography showed 92:8ratio of isomers. A third recrystallization from ethyl acetate (10 mL)afforded yellow needles; 1.25 g. mp=152-154° C. Gas chromatographyshowed 96:4 isomer ratio. Proton NMR confirmed as the desireddialdehyde. GC-MS analysis was consistent with the desired dialdehyde,showing a prominent M⁺ ion at 164 m/z and a base ion at 91 m/z.

The ethyl acetate filtrate was combined with the yellow solid from themethanol filtrate (crop 4) and concentrated on a rotary evaporator togive a yellow solid; 6.0 g. Gas chromatography showed a 53:34 mixture ofthe two isomers along with other impurities.

The solid was dissolved in 100 mL dichloromethane and Davisil grade 643silica gel (33.5 g) was added. The mixture was stripped on a rotaryevaporator at 35° C. The silica gel with adsorbed material was thenadded to the sample introduction module for the Biotage system, whichalready contained a plug of glass wool and a layer of sand. The silicagel was then topped with filter paper. The Biotage 75S column waspreviously wetted with the solvent mixture with a radial compression of35 psi and solvent pressure of 20 psi. The column was eluted with 85:15hexane:ethyl acetate (6,000 mL). A void volume of 1,000 mL including theprewet stage was taken. Fractions of 250 mL were collected and combinedbased on thin layer chromatogram analysis. These fractions wereconcentrated on a rotary evaporator at 35° C. as shown below. FractionContent Appearance Amt (g) Comment 1 blank 2-3 A 4 tr A  5-10 B yellowsolid 3.9 Product Cut 11-18 tr B or tr C No evidence of close elutingimpurity 19-20 tr B or C & D

Fractions 5-10: The yellow solid was slurried in hexane and suctionfiltered to give a bright yellow solid; 2.5 g. Gas chromatography showedan mixture of dialdehyde isomers in a ratio of 67:33.

Total yield of 96-97% E,E,E-dialdehyde=10.75+1.25=12.0 g (58.8% yield).

Isomerization of 2,7-Dimethyl-2,4,6-ocatrienedial withpara-Toluenesulfinic Acid

Under a nitrogen atmosphere, the 2:1 isomeric mixture of2,7-dimethyl-2,4,6-ocatrienedial and its off-isomer (2.5 g; 15.2 mmol)and 4-toluenesulfinic acid (0.35 g; 2.2 mmol) and 50 mL anhydrous1,4-dioxane was heated at reflux for 15 minutes. An aliquot (7 drops)was diluted in 0.5 mL 4:1 ether:dichloromethane and dried over K₂CO₃.Gas chromatography showed a 91:9 mixture of desired isomer andoff-isomer.

After cooling overnight at room temperature, the resulting slurry wasdissolved in 100 mL 4:1 ether:dichloromethane and washed successivelywith water (50 mL×3), 0.2M aqueous NaOH (50 mL), water (50 mL×2) andsaturated brine (50 mL×3). After separation of the layers, the remainingrag layer was dissolved in dichloromethane. The combined organic layerswere dried over MgSO₄, filtered and concentrated on a rotary evaporatorat 40° C. to give an orange solid; 2.2 g. Gas chromatography showed 93:7ratio of desired dialdehyde to off-isomer. This solid was slurried inhexane and suction filtered to give an orange solid; 2.15 g. This solidwas recrystallized from 20 mL refluxing ethyl acetate by cooling to30-40° C. and then in the freezer for 1 hr. The slurry was suctionfiltered and washed with freezer-chilled ethyl acetate to giveyellow-orange needles; 1.65 g. mp=158-160° C. lit mp=161-162° C.

Gas chromatography showed 96:4 ratio of desired dialdehyde tooff-isomer. Proton NMR and Carbon NMR were consistent with the desireddialdehyde isomer.

Yield=66%

Scaleup Prep of Methyl Tiglate with Thionyl Chloride in Methanol

A mechanically stirred solution of tiglic acid (397.35 g; 3.97 mol) in3,000 mL methanol was treated dropwise with neat thionyl chloride (397mL; 5.44 mol) over a period of 130 minutes as the temperature climbedfrom 14° C. to a maximum of 50° C. after 80 minutes with no externalcooling. Gas chromatography of an aliquot showed complete conversion tothe ester with no detectable tiglic acid. After stirring at ambienttemperature for 1 hr, the solution was distilled at atmospheric pressurethrough a silvered, vacuum jacketed Vigreux column (400 mm×20 mm). Thecondensate was collected at mainly 57-61° C. with a pot temperature of58-63° C.; 630 mL in 2 hrs. Gas chromatography showed significant methylester in the distillate.

The Vigreux column was swapped with a less efficient column (30×2 cmw/less indentations) to speed up the rate of distillation. At a pottemperature of 69-71° C., distillate was collected with a headtemperature of 65-69° C.; 1,300 mL over 2.25 hrs.

Gas chromatography showed significant methyl ester in the distillate.The atmospheric distillation was continued until the pot temperaturereached 87° C., distillate was collected during this period at a headtemperature of 69-83° C.; 975 mL over 2 hrs. Gas chromatography showedsignificantly more methyl ester in the distillate than earlierfractions.

The yellow two-phase mixture in the pot was extracted with ether (300 &200 mL), dried over K₂CO₃, filtered and concentrated on a rotaryevaporator at 25° C. to give an orange oil; 132.6 g (29.3% yield). Gaschromatography showed product. Proton NMR and carbon NMR were consistentwith the desired product with trace ethyl ether. Gas chromatography ofthe ether condensate showed some methyl ester in the overheads.

Distillate 3: The third methanol distillate (975 mL) was concentrated onthe rotary evaporator at 25° C. to give a two phase mixture (100-150mL). This mixture was extracted with ether (100 & 50 mL), dried overK₂CO₃.

Distillate 2: The second methanol distillate (1,300 mL) was concentratedon the rotary evaporator at 25° C. to give a two phase mixture (30-50mL). This mixture was extracted with ether (2×50 mL), dried over K₂CO₃.

The concentrated ether extracts for distillate 2 and distillate 3 werecombined, suction filtered and concentrated on a rotary evaporator at25° C. to give a colorless oil; 77.3 g.

Proton NMR and carbon NMR matched previous spectra of the desired methylester.

Total Yield=132.6+77.3=209.9 g (46.3%)

Alternatively, 1) methyl tiglate is commercially available from Alfa,Lancaster or Acros. and 2), pilots can be run to make phosphonium saltvia JOC, 64, 8051-8053 (1999).

Bromination of Methyl Tiglate

A mechanically stirred slurry of methyl tiglate (209.9 g; 1.84 mol) andN-bromosuccinimide (327.5 g; 1.84 mol), 70% benzoyl peroxide (3.2 g;0.009 mol) in 2,000 mL carbon tetrachloride was heated to reflux (78-81°C.) with a 1 L Kugelrohr bulb between the 5 L reaction flask and thereflux condenser. After 2 hrs, reflux was stopped, the mantle droppedand the stirrer shutoff. All of the solids floated on the CCl₄ solution,suggesting succinimide with negligible NBS. The slurry was cooled in anice bath to 20° C. and suction filtered to give an offwhite solid; 180.7g. No wash. The yellow filtrate was washed with water (1 L×3), driedover MgSO₄. Gas chromatography showed starting methyl tiglate and thetwo monobromides in 1:2:1 ratio along with other minor components.

After filtering off the MgSO₄, the light yellow filtrate wasconcentrated on a rotary evaporator at 35° C. to give a light yellowoil; 327.1 g. Proton NMR and gas chromatography suggested the followingcomposition: Component NMR (mole %) GC (Area %) γ-Bromo 50% 49% α-Bromo26% 21% α,γ-Dibromo (?)  7%  4% Methyl Tiglate  6% 10% Other 11% —

Yield of desired product adjusted for 50% assay=46.0%

This oil is used as is in the next step.

Scaleup Reaction of Methyl γ-Bromotiglate with Triphenylphosphine inAcetonitrile with Slightly Higher Payload

Under a nitrogen atmosphere in a 5 L, 4-neck flask, the crude mixture ofmethyl γ-bromotiglate (322.6 g; 85% allylic bromide; 1.42 mol) in 1,300mL anhydrous acetonitrile was stirred mechanically.

A solution of triphenylphosphine (387.0 g; 1.48 mol) in 2,000 mL ethylacetate was added dropwise over a period of 4 hours. During theaddition, the temperature climbed from 22° C. to a maximum of 30° C.after adding about 40% in the first 75 minutes. After adding 60% of thetriphenylphosphine solution over 120 minutes, the solution became cloudyand continued to precipitate solids through the rest of the addition.After the addition, the funnel was rinsed with ethyl acetate (600 mL)and chased into the reaction mixture. The cream slurry was stirred atambient temperature over the weekend.

The white slurry was suction filtered and the cake was washed with 2:1ethyl acetate:acetonitrile (150 mL×3). The white solid (352.55 g) wasdried in a vacuum oven at 40° C. for 4 hrs (constant weight after 2hrs); 322.55 g. mp=187-188° C. (dec). lit mp=183° C. (dec). Proton NMRand Carbon NMR matched previous spectra for the desired phosphoniumsalt. LC-MS analysis showed one major component, whose electrospray massspectrum in the positive mode was consistent with the desiredphosphonium salt giving a molecular ion at 375 m/z. Phosphorus NMRshowed a single phosphorus signal at 22.0 ppm.

Yield based on starting methyltiglate=100×322.55/(455.32×1.84×322.6/327.1)=39.0%

Prep of (3-Carbomethoxy-2-Z-buten-1-ylidene)triphenylphosphorane

A mechanically stirred slight slurry of(3-carbomethoxy-2-E-buten-1-ylidene)triphenylphosphonium bromide (154.8g; 0.34 mol) in 3,400 mL deionized water was treated dropwise with asolution of sodium hydroxide (13.6 g; 0.34 mol) in 500 mL water at 23°C. over a period of 32 minutes with no obvious exotherm, but immediateprecipitation of a bright yellow solid. After stirring for 15 minutes,the bright yellow slurry was suction filtered, washed with water (1,500mL) and sucked dry to give a canary yellow solid; 151.7 g. This solidwas dried in a vacuum oven at 35-45° C. (3:50 pm) overnight.

After drying in the vacuum oven at 35-45° C. for 22.5 hrs, a constantweight was obtained; 107.8 g. mp=144-160° C. lit mp=145-165° C. ProtonNMR was similar to the previous spectrum of the desired ylideconsidering the differences in NMR field strength. Carbon NMR showed themethyl carbon's at 50.2 and 11.8 ppm with a complex aromatic region andno obvious signals for the olefinic carbons and the ylide carbon.

Yield=84.7%

Pilot Prep of Dimethyl Crocetinate

Under a nitrogen atmosphere, a magnetically stirred mixture of(3-carbomethoxy-2-Z-buten-1-ylidene)triphenylphosphorane (12.8 g; 34.2mmol) and 2,7-dimethyl-2,4,6-ocatrienedial (2.1 g; 12.8 mmol) in benzene(128 mL) was heated to reflux for 6 hrs using a timer.

The resulting slurry was cooled in an ice bath for 40 minutes, suctionfiltered, washed with benzene and sucked dry to melt the frozen benzeneto give a red solid; 2.1 g. TLC analysis showed a single, yellow spot.This solid was dried in a vacuum oven at 40-45° C. for 70 minutes; 1.85g (40.5% yield). uncorrected mp=210-213° C. lit³ mp=214-216° C. ProtonNMR was similar to the previous spectrum of dimethyl crocetin on 90 MHzinstrument. Carbon NMR showed all 11 unique carbon signals with thecorrect chemical shift for the desired dimethyl ester with one minorimpurity signal that may be residual benzene. Electrospray mass spectrumsuggested decomposition and recombination of fragments.³ E. Buchta & F. Andree, Chem Ber, 93, 1349 (1960).

TLC analysis showed that the red filtrate contained additional product,triphenylphosphine oxide and an orange component with an R_(f) slightlylower than the isolated solid. The red filtrate was concentrated on arotary evaporator at 35° C. to give red solids; 13.2 g. This solid washeated at reflux in methanol (25 mL). The resulting slurry was thencooled in an ice bath, suction filtered after 60 minutes and washed withmethanol to give a red solid; 0.6 g. This solid was dried in the vacuumoven at 45° C. 135 minutes; 0.5 g. mp=203-208° C. Proton NMR showeddesired diester with residual impurities. Carbon NMR showed only signalsfor desired product. TLC analysis showed streaky product spot.

Filtrate was concentrated and saved.

Second Prep of Dimethyl Ester of Crocetin

Under a nitrogen atmosphere, 2,7-dimethyl-2,4,6-ocatrienedial (11.95 g;12.8 mmol) was added in one portion to a mechanically stirred slurry of(3-carbomethoxy-2-Z-buten-1-ylidene)triphenylphosphorane (73.0 g; 195.0mmol) in 400 mL benzene and then chased with 330 mL benzene. Theresulting brown slurry was heated to reflux for 6 hrs using a timer andcooled to room temperature overnight under nitrogen.

The resulting slurry was cooled in an ice bath to 6-10° C., suctionfiltered and washed with benzene (50 mL×2) to give a red solid; 10.05 g.TLC analysis showed a single yellow spot. This solid was dried in avacuum oven at 40° C. (9:00 am) for 3.5 hrs with no weight loss; 10.05 g(38.7% yield). mp=211-214° C. lit mp=mp=214-216° C. Proton NMR andCarbon NMR matched the previous spectra for the desired dimethyl esterof crocetin.

The red filtrate was concentrated on a rotary evaporator at 40° C. togive a red solid; 84.4 g. TLC analysis was similar to the pilot run.This solid was slurried in 165 mL methanol at reflux with magneticstirring. The resulting slurry was then cooled in an ice bath for 2.5hrs, suction filtered and washed with a minimal amount of methanol togive an orange paste; 10.5 g. TLC analysis showed a single, yellow spot.This paste was dried in a vacuum oven at 45° C. for 190 minutes; 5.6 g.mp=201-208° C. NMR showed desired diester with unknown aromaticimpurities.

This impure solid and two other similar solids from earlier runstotaling 6.5 g were dissolved in refluxing chloroform (75 mL) anddiluted with methanol and cooled in the refrigerator overnight.

The slurry was suction filtered and washed with a minimal amount ofmethanol to give red crystalline solid; 6.1 g. This solid was dried inthe vacuum oven at 45° C. for 3 hrs until constant weight; 4.25 g.mp=211-213° C. Proton NMR and carbon NMR showed other olefinic oraromatic impurities. The solid was dissolved in refluxing toluene (150mL) and eventually cooled in the refrigerator for 130 minutes. Theslurry was suction filtered and washed with toluene to give a red solid;2.05 g. This solid was dried in the vacuum oven at 45° C. for 50 minuteswith no weight change; 2.05 g. mp=214-216° C. Proton NMR showed thedesired dimethyl crocetin with some residual toluene and negligibleoff-isomer impurities. Carbon NMR showed the desired dimethyl crocetinwith no detectable off-isomer impurities and 2-3 new residual signalsthat were consistent with toluene. Yield=45.5%.

Prep of Disodium Salt of Crocetin

A mechanically stirred slurry of dimethyl crocetin (13.95 g; 39.1 mmol)and 40 wt % aqueous sodium hydroxide (273 mL; 3.915 mol) and methanol(391 mL) was heated at reflux at 74° C. for 12 hrs using a timer.

The orange slurry was suction filtered through a Buchner funnel withfilter paper and a sintered glass funnel. Slow filtration.⁴ The slurryin the sintered glass funnel was added to the solids in the Buchnerfunnel. The orange paste was washed with water (100 mL×3) and then withmethanol (50 mL×3). The orange paste was dried in a vacuum oven at45-50° C.⁴ Filtered faster through sintered glass until the filter clogged afterdrying out. However, water wash unclogged the filter.

After 21 hrs, the orange clumps weighted 24.25 g. The material waspulverized with a spatula and dried in the vacuum oven at 45-50° C.

After a total of 65.5 hrs of drying, amount of orange powder was 23.1 g.The infrared spectrum showed extra bands compared to the reported IRspectrum of TSC, especially large bands at 3424 and 1444 cm⁻¹. ProtonNMR showed no evidence of methyl esters. However, the integration of theolefinic and methyl regions were off, possibly due to phasing problems.

Assuming that the excess weight was due to sodium hydroxide, the orangesolid was stirred magnetically in 400 mL deionized water for 35 minutes.The slurry was suction filtered and the cake washed with deionized water(50 mL×2) to give an orange paste. This material was dried in a vacuumoven at 45-50° C. until constant weight. After about 7 hrs, the solidwas crushed and pulverized and dried further in the vacuum oven at 45°C. overnight.

After 21 hrs of drying at 45° C., amount of solid was 13.25 g. Afterfurther pulverizing and drying in the vacuum oven at 45° C., amount ofsolid was 13.15 g. The infrared spectrum was consistent with thereported IR spectrum. Proton NMR gave a proton NMR spectrum that wasconsistent with The disodium salt. HPLC analysis showed one majorcomponent with possibly one minor impurity. The electrospray negativeion mass spectrum of the major component was consistent with the desireddisodium salt of crocetin. Carbon NMR showed all ten unique carbonsignals for disodium salt of crocetin, verifying the symmetry of themolecule.

The original filtrate of water, sodium hydroxide and methanolprecipitated more solids during the water wash. This slurry was suctionfiltered, washed with water to give an orange paste. This paste wasdried in the vacuum oven at 45° C. for 18.5 hrs to give an orange solid;0.65 g. The spectral data were consistent with the desired disodium saltof crocetin. This solid was combined with the first crop.

Yield=13.15+0.65=13.8 g (94.8%).

Elemental Analyses of the first crop showed unacceptable values for thedesired product, suggesting sodium hydroxide contamination of thedisodium salt of crocetin.

Water Wash of Disodium Salt of Crocetin

The disodium salt of crocetin (13.6 g) was slurried in 150 mL deionizedwater and stirred magnetically at room temperature for 1 hr. The slurrywas suction filtered onto a Buchner funnel. The orange paste was thenwashed with water and the pH of the orange filtrate monitored.

The orange paste was sucked dry on the filter with a rubber dam. Thispaste was dried in a vacuum at 25-55° C. for 5.5 hrs to give a friableorange solid; 11.2 g. This solid was pulverized, transferred to a bottleand dried in the vacuum oven at 45° C. overnight.

Amount=11.1 g. Recovery=81.6%. The IR and Proton NMR spectra matchedprevious IR and proton NMR spectra of the desired disodium salt ofcrocetin. HPLC analysis showed a single component at 420 nm, whoseelectrospray mass spectrum in the negative ion mode was consistent withcrocetin.

Carbon NMR showed all ten unique carbon signals with the correctchemical shifts for the desired disodium salt of crocetin. Elementalanalysis gave acceptable data for the desired product.

REFERENCES

-   1. Tetrahedron Letters, 27, 4983-4986 (1986).-   2. F. J. H. M. Jansen, M. Kwestro, D. Schmitt & J. Lugtenburg, Rec.    Trav. Chem. Pays-Bas, 113, 552-562 (1994) and references cited    therein.-   3. J. H. Babler, U.S. Pat. No. 4,107,030, Apr. 21, 1992.-   4. T. W. Gibson & P. Strassburger, J. Org. Chem., 41, 791 (1976)    & J. M. Snyder & C. R. Scholfield, J. Am. Oil Chem. Soc., 59, 469    (1982).

Example 6

Purity Determination of TSC Made According to the Improved SynthesisMethod

For the TSC material synthesized according to the method of Example 5,the ratio of the absorbance at 421 nm to the absorbance at 254 nm was11.1 using a UV-visible spectrophotometer.

Example 7

Oral Administration of TSC

TSC has been shown, in rats, to be absorbed into the blood stream whenadministered orally (via a gavage technique). In two rats, it was foundthat 1 to 2% of the dosage given was present in the blood stream at atime of 15 to 30 minutes after being given. The maximum amount absorbedorally actually occurred earlier than that time.

It will be readily apparent to those skilled in the art that numerousmodifications and additions can be made to both the present compoundsand compositions, and the related methods without departing from theinvention disclosed.

1-25. (canceled)
 26. A method of synthesizing a BTCS compound having theformulaYZ-TCRO-ZY where: Y=a cation Z=a polar group which is associated withthe cation, and TCRO=trans carotenoid skeleton, comprising the steps of:a) coupling a symmetrical dialdehyde containing conjugated carbon-carbondouble bonds with a triphenylphosphorane, b) saponifying the product ofstep a) to yield said BTCS compound.
 27. A method as in claim 26 whereinthe coupling of step a) is made using[3-carbomethoxy-2-buten-1-ylidene]triphenylphosphorane.
 28. A method asin claim 26 wherein the product of step a) is saponified using asolution of NaOH and methanol.
 29. A method as in claim 26 wherein afterstep a) is the step of isolating the desired product of the couplingreaction.
 30. A method of saponifying a symmetrical diester containingconjugated carbon-carbon double bonds to form a BTCS, comprising thesteps of: a) solubilizing the symmetrical diester containing conjugatedcarbon-carbon double bonds with a compound selected from the groupconsisting of methanol, ethanol, propanol and isopropanol, and b) mixingthe solution of step a) with a base to form said BTCS.
 31. A method asin claim 30 wherein the base is selected from the group consisting ofNaOH, KOH, and LiOH.
 32. A method as in claim 30 wherein the diester issaponified using methanol and NaOH. 33-59. (canceled)
 60. A BTCScompound synthesized by: a) coupling a symmetrical dialdehyde containingconjugated carbon-carbon double bonds with a triphenylphosphorane, b)saponifying the product of step a) to yield said BTCS compound, whereinsaid compound has the structure:YZ-TCRO-ZY where: Y=a cation Z=polar group which is associated with thecation, and TCRO=trans carotenoid skeleton having pendant groups X,wherein the pendant groups X, which can be the same or different, are alinear or branched group having 10 or less carbon atoms, or a halogen,and wherein said compound is not TSC or a norbixin salt.